The World Health Organization recognizes inorganic arsenic as a carcinogen, and a serious threat to millions of people. One mechanism cells use to deal with exposure to toxicants such as arsenic, is to pump them out of the cytoplasm using a special type of ABC transporter called a multidrug resistance protein (MRP). The yeast MRP, YCF1, actively pumps glutathione:arsenite conjugates into the vacuole, resulting in arsenic resistance. A YCF1 deletion strain lacks pump activity and is hypersensitive to arsenic. We propose that enhanced versions of YCF1 will confer increased arsenic uptake, accumulation, and resistance upon transgenic cells, and the Aycfl deletion strain will provide a convenient experimental system to test this hypothesis. The goal of this work is to identify conserved residues and domains within MRPs responsible for regulating arsenic toxicity, and will be addressed with the following specific aims: (1) Enhance the arsenic- transporting pump activity of YCF1. We will use PCR to randomly mutagenize the YCF1 gene, homologous recombination with a yeast expression vector to generate a library of mutants in a Aycfl yeast background, and phenotypic screening to identify mutations conferring enhanced arsenic resistance on Aycfl cells. (2) Explore the effects of corresponding mutations on human and plant YCF1 homologs. Human (MRP1, 2) and plant (AtMRPI, 3) multidrug resistance genes will be introduced into Aycfl yeast, and tested for their ability to rescue the Aycfl arsenic-sensitivity. Promising mutations identified in YCF1 will be introduced into the corresponding sites of these human and plant MRPs by site-directed mutagenesis, and the ability of these mutant MRPs to complement Aycfl yeast with enhanced arsenic pump activity will be examined. (3) Demonstrate an MRP-mediated reduction in arsenic-induced toxicity. Independent plant and CHO (Chinese hamster ovary) cell lines carrying the most promising mutant MRP versions identified will be generated and examined for acquisition of enhanced arsenic pump activity, measured by cell survival rates, arsenic accumulation, and induction of stress-related transcripts. Our coordinated work with yeast, plant, and human MRPs, in three different biochemical backgrounds, will define evolutionarily conserved regions that are important for arsenic pump activity and/or substrate specificity. RELEVANCE: This information will contribute toward dissecting the functions of the entire MRP subfamily of ABC transporters, several members of which have been implicated in human diseases, resistance to chemotherapeutic drugs, or metal toxicity. In addition, because YCF1 activity contributes not only to arsenic, but also to cadmium, mercury, and lead resistance in yeast, our results will likely be applicable toward several environmental toxicants.